Towards Improved Accuracy of Bordetella pertussis Nucleic Acid Amplification Tests

Abstract

In many clinical microbiology laboratories, nucleic acid amplification tests such as PCR have become the routine methods for the diagnosis of pertussis. While PCR has greatly increased the ability of laboratories to detect Bordetella pertussis infections, it has also been associated with false-positive results that can, given the tendency of B. pertussis to cause outbreaks, result in unnecessary and costly control measures. The species specificity of Bordetella gene targets and their number of copies per genome greatly impact the performance characteristics of nucleic acid amplification tests for B. pertussis . It is crucial that laboratorians recognize these characteristics, to limit false-positive test results and prevent pseudo-outbreaks.

Evaluation of a Multitarget Real-Time PCR Assay for Detection of Bordetella Species during a Pertussis Outbreak in New Hampshire in 2011

“…Four specimens displayed 35 Յ C T Ͻ 36 for IS481, and two each were positive by ptxS1 and culture. No specimens with an IS481 C T of Ն36 tested positive by ptxS1 assay, as IS481 is present at a much higher copy number (more than 50 copies per genome) in B. pertussis than ptxS1 (a single copy per genome) (10,11). However, one specimen with an IS481 C T value of 38.2 was ptxS1 negative and culture positive.…”

Implementation and Assessment of the Use of Real-Time PCR in Routine Diagnosis for Bordetella pertussis Detection in Brazil

“…This sequence is often the target of choice because it is found in multiple copies in the genome of B. pertussis (50-238 copies per genome), making this test much more sensitive. However, because of its high copy number, false positive results may be generated and positive results using only a target may lead to a false diagnosis of pertussis (15,22,23). The IS481 is also present in B. holmesii (8-10 copies per genome) and in B. Bronchiseptica, but the presence of IS481 is host dependent in this specie and is found in several animals, being rare human isolates (24).…”

“…A positive test for IS481 requires additional testing, using assays that detect toxin gene, pertactin or other targets specific to B. pertussis, may be useful in interpreting the results for confirmation of the species. An isolated result for IS481 should be reported as negative or inconclusive for B. pertussis if there isn´t possibility of a target additional (13,15,25).…”

“…Several PCR protocols have been developed in recent years for different targets in the genome of B. pertussis like an insertion sequence IS481, pertussis toxin gene promoter (ptxA-Pr), pertussis toxin S1 subunit (ptxS1), porin gene, pertactin filaments hemagglutinin gene and adenylate cyclase (14,15,20,21). Many laboratories use the insertion sequence IS481 in PCR assays to determine the presence of B. pertussis DNA.…”

“…The high sensitivity (70-90%) and specificity (86-100%), along with a shorter response time to results, low risk of contamination and ease of performance, makes RT-PCR an alternative method for the diagnosis of many diseases, among them, the pertussis (13)(14)(15).…”